Summary of work: GADD153 is a highly conserved mammalian gene whose expression is increased in response to a variety of stresses. It is a member of the CCAAT/enhancer-binding protein C/EBP) family of transcription factors and has been implicated in both growth arrest and apoptosis. However, its role in these processes is far from clear. Studies in this project have focused on the regulation of GADD153 gene expression and the function of the GADD153 protein during the cellular response to diverse stimuli. With respect to regulation, recent efforts have concentrated in two areas. First, we have examined the role of the CREB-family transcription factor ATF6 (implicated in regulating gene expression during the response to stress to the endoplasmic reticulum (ER stress) in regulating GADD153 transcription. We have provided evidence that overexpression of ATF6 transactivates the GADD153 promoter and that antisense ATF6 attenuates GADD153 expression by ER stress. Current studies are aimed at further characterizing the role of ATF6 in regulating GADD153 expression by other stresses. Second, we are examining the possible role of PLCgamma1 in regulating GADD153 expression. We have noted that PLCgamma1 deficient cells show greatly diminished expression of GADD153 during various stress conditions. Current studies are attempting to understand the basis for this phenomenon, particularly whether PLCgamma1 serves as an upstream regulator of GADD153 transcriptional activity. Studies on the function of GADD153 have centered on its ability to influence cell survival during stress. Employing several models in which the GADD153 protein is over-expressed, we have found that elevated GADD153 heightens the cell's sensitivity to a variety of stressful treatments resulting in apoptosis. GADD153 overexpression also leads to a reduction in cellular glutathione levels, elevated amounts of reactive oxygen species and reduced levels of Bcl-2 protein. Current studies are further exploring the association between these phenomena and the reduced survival of GADD153-expressing cells. We are also investigating the molecular mechanism whereby GADD153 represses Bcl-2 expression. Our findings suggest that GADD153 serves a pro-apoptotic function through perturbations in cellular redox status.